Disclaimer: The manuscript and its contents are confidential, intended for journal review purposes only, and not to be further disclosed. URL: https://circres-submit.aha-journals.org Manuscript Number: CIRCRES/2018/313854DR1 Title: ATF6 regulates cardiac hypertrophy by transcriptional induction of the mTORC1 activator, Rheb Authors: Christopher Glembotski Erik Blackwood Christoph Hofmann Michelle Santo Domingo Alina Bilal Winston Stauffer Donna Thuerauf Fred Kolkhorst Oliver Müller Tobias Jakobi Christoph Dieterich Hugo Katus Shirin Doroudgar Anup Sarakki Adrian Arrieta For Circulation Research Peer Review. Do not distribute. Destroy after use. Circulation Research Online Submission: https://circres-submit.aha-journals.org Circulation Research Homepage: http://circres.ahajournals.org Circulation Research Editorial Office 3355 Keswick Rd, Main Bldg 103 Baltimore, MD 21211 [email protected] Telephone: 410-327-5005 1 2 CIRCRES/2018/313233D-A 3 4 ATF6 regulates cardiac hypertrophy by transcriptional induction of the mTORC1 5 activator, Rheb 6 7 Erik A. Blackwood, B.S. 1, Christoph Hofmann1,2,3, Michelle Santo Domingo, B.S. 8 1, Alina S. Bilal, B.A. 1, Anup Sarakki, B.S. 1, Winston Stauffer, B.S.1, Adrian 9 Arrieta, M.S.1, Donna J. Thuerauf, M.S.1, Fred W. Kolkhorst, Ph.D.1, 10 Oliver J. Müller2,3,4, M.D., Tobias Jakobi3,5, Ph.D., 11 Christoph Dieterich3,5, Ph.D., Hugo A. Katus, M.D. 2,3, Shirin Doroudgar, Ph.D.2,3 12 and Christopher C. Glembotski, Ph.D. 1* 13 14 15 1San Diego State University Heart Institute and the Department of Biology, San 16 Diego State University, San Diego, CA 92182 17 2Department of Cardiology, Angiology, and Pneumology, University Hospital 18 Heidelberg, Innere Medizin III, Im Neuenheimer Feld 669, 19 69120 Heidelberg, Germany. 20 3DZHK (German Centre for Cardiovascular Research), Partner Site 21 Heidelberg/Mannheim, 69120 Heidelberg, Germany. 22 4 Department of Internal Medicine III, 23 University of Kiel, Arnold-Heller-Str.3, Kiel, Germany 24 5Section of Bioinformatics and Systems Cardiology, Department of Internal 25 Medicine III, Klaus Tschira Institute for Integrative Computational Cardiology, 26 University Hospital Heidelberg, Heidelberg, Germany 27 28 29 Short Title: ATF6 regulates cardiac growth 30 *Corresponding Author: Christopher C. Glembotski, the SDSU Heart Institute and 31 Department of Biology, San Diego State University, 5500 Campanile Drive, San Diego, 32 CA 92182, USA. Tel.: (619) 594-2959; FAX: (619) 594-5676; E-mail: 33 [email protected] 34 35 36 For Circulation Research Peer Review. Do not distribute. Destroy after use. 1 1 ABSTRACT: 2 Rationale: ER stress dysregulates ER proteostasis, which activates the transcription 3 factor, ATF6, an inducer of genes that enhance protein folding and restore proteostasis. 4 Due to increased protein synthesis, it is possible that protein folding and, thus, ER 5 proteostasis are challenged during cardiac myocyte growth. However, it is not known 6 whether ATF6 is activated, and if so, what its function is during hypertrophic growth of 7 cardiac myocytes. 8 Objective: To examine the activity and function of ATF6 during cardiac hypertrophy. 9 Methods and Results: We found that ATF6 was activated and ATF6-target genes were 10 induced in mice subjected to an acute model of trans-aortic constriction (TAC), or to 11 free-wheel exercise, which promote adaptive cardiac myocyte hypertrophy with 12 preserved cardiac function. Cardiac myocyte-specific deletion of Atf6 (ATF6 cKO) 13 blunted TAC- and exercise-induced cardiac myocyte hypertrophy and impaired cardiac 14 function, demonstrating a role for ATF6 in compensatory myocyte growth. Transcript 15 profiling and chromatin immunoprecipitation identified RHEB as an ATF6-target gene in 16 the heart. RHEB is an activator of mTORC1, a major inducer of protein synthesis and 17 subsequent cell growth. Both TAC and exercise upregulated RHEB, activated mTORC1, 18 and induced cardiac hypertrophy in WT mouse hearts, but not in ATF6 cKO hearts. 19 Mechanistically, knockdown of ATF6 in neonatal rat ventricular myocytes blocked 20 phenylephrine (PE)-, and insulin-like growth factor 1 (IGF1)-mediated Rheb induction, 21 mTORC1 activation, and myocyte growth, all of which were restored by ectopic RHEB 22 expression. Moreover, AAV9-RHEB restored cardiac growth to ATF6 cKO mice 23 subjected to TAC. Finally, ATF6 induced RHEB in response to growth factors, but not in 24 response to other activators of ATF6 that do not induce growth, indicating that ATF6 25 target gene induction is stress-specific. 26 Conclusions: Compensatory cardiac hypertrophy activates ATF6, which induces Rheb 27 and activates mTORC1. Thus, ATF6 is a previously unrecognized link between growth 28 stimuli and mTORC1-mediated cardiac growth. 29 30 Key Words: 31 Myocytes, cardiac protein folding, proteostasis, cardiac hypertrophy, ATF6, Rheb, 32 mTORC1 33 For Circulation Research Peer Review. Do not distribute. Destroy after use. 2 1 Non-standard Abbreviations and Acronyms 2 3 AAV adeno-associated virus 4 AdV adenovirus 5 ANOVA analysis of variance 6 ATF6 activating transcription factor 6 alpha 7 ATF6 cKO ATF6 alpha conditional knockout 8 Cat Catalase 9 ER endoplasmic reticulum 10 Grp78 78 kilodalton glucose-regulated protein, Hapa5 11 HR heart rate 12 HW heart weight 13 ICF immunocytofluorescence 14 LV left ventricle 15 LVEDV left ventricular end diastolic volume 16 LVESV left ventricular end systolic volume 17 LVIDD left ventricular inner diameter in diastole 18 LVIDS left ventricular inner diameter in systole 19 PWTD left ventricular posterior wall thickness in diastole 20 PWTS left ventricular posterior wall thickness in systole 21 Rheb Ras homologue enriched in brain 22 SR sarcoplasmic reticulum 23 TAC transverse aortic constriction 24 TL tibia length 25 TM tunicamycin 26 UPR unfolded protein response 27 28 For Circulation Research Peer Review. Do not distribute. Destroy after use. 3 1 INTRODUCTION: 2 3 Protein homeostasis, or proteostasis involves the coordination of protein synthesis 4 and folding to ensure proteome integrity and vital cell function1. In cardiac myocytes the 5 endoplasmic reticulum (ER) is a major site of synthesis of proteins that are critical for 6 proper function of the heart, including many calcium-handling proteins, receptors, and 7 secreted proteins, such as hormones, stem cell homing factors, and growth factors2, 3. 8 Therefore, ER proteostasis maintains the integrity of the cardiac myocyte proteome and, 9 thus, cardiac contractility. Increased protein synthesis in growing cardiac myocytes must 10 be balanced by increased protein-folding to avoid the accumulation of toxic misfolded 11 proteins; thus growth poses a potential challenge to cardiac myocyte proteostasis4. 12 However, the molecular mechanisms underlying the maintenance of ER proteostasis 13 during cardiac myocyte growth are not well understood. 14 ER proteostasis is controlled in all mammalian cells by several ER-transmembrane 15 sensors of protein misfolding, including the adaptive transcription factor, ATF65. When 16 protein synthesis surpasses the capacity of the protein-folding machinery, increases in 17 misfolded proteins cause the translocation of the ER-transmembrane, 670-amino acid, 18 90 kD form of ATF6, to the Golgi, where it is clipped, liberating an N-terminal fragment 19 that serves as a transcription factor. This 50 kD active form of ATF6 regulates a gene 20 program that is responsible for the expression of numerous proteins that enhance ER 21 protein folding, which adaptively restores the balance between protein synthesis and 22 folding6, 7. Thus, nodal proteostasis regulators, such as ATF6, that sense and maintain 23 this balance could play important roles in optimizing cardiac myocyte growth; however, 24 neither the activation nor the function of ATF6 in the setting of hypertrophic cardiac 25 growth has been examined. Accordingly, here we studied the effects of Atf6 deletion in 26 mouse hearts and in cultured cardiac myocytes during physiologically-relevant 27 maneuvers known to promote compensatory cardiac hypertrophy in either a concentric 28 (pressure overload)8 or eccentric (exercise)9 manner, positing that the absence of ATF6 29 would imbalance proteostasis, which would be maladaptive. 30 Both growth maneuvers activated ATF6, and Atf6 deletion was maladaptive, as 31 evidenced by impaired cardiac function. However, surprisingly, cardiac myocyte growth 32 was also impaired upon Atf6 deletion. This was unexpected, since Atf6 is not known to 33 be required for cardiac myocyte growth. Further mechanistic studies showed that ATF6 34 is activated as a result of the increased demands placed on the ER protein folding 35 machinery during growth-related increases in protein synthesis. Moreover we found that 36 Atf6 serves a previously unrecognized role as a molecular link between growth stimuli 37 and activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a 38 major promoter of protein synthesis and consequent growth of cardiac myocytes10-15. 39 Two conditionsFor Circulation need to be met forResearch mTORC1 to be Peer activated; Review. 1) in resp onseDo tonot a growth 40 stimulus, mTORC1 needs to translocate to organelles, such as lysosomes16, where it 41 encounters the small GTPase distribute. activator of Destroy mTORC1, Rheb after17, and use. 2) Rheb must be 42 active and present in sufficient quantities18. In terms of Rheb activation, it is known that 43 growth stimuli lead to the phosphorylation and, thus, inhibition of the Rheb GTPase- 44 activating protein (GAP), TSC1/TSC219, which increases the GTP-loading state and, 45 thus, the activity of Rheb. However, the molecular mechanisms underlying the Rheb 46 gene expression are less well understood. Here, we showed, for the first time, that ATF6 47 is an inducer of Rheb, and in this way, ATF6 coordinates protein synthesis and protein 48 folding, ensuring the adaptive maintenance of proteostasis in growing cardiac myocytes. 49 Thus, ATF6 is a newly identified and essential member of mTORC1 growth signaling in 50 cardiac myocytes in the heart. 4 1 Methods: 2 Further details on the Methods can be found in the Online Supplement.